Structure of the DDB1-CRBN E3 Ubiquitin Ligase in Complex with Thalidomide
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Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Fischer, E. S., K. Böhm, J. R. Lydeard, H. Yang, M. B. Stadler, S. Cavadini, J. Nagel, et al. 2015. “Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.” Nature 512 (7512): 49-53. doi:10.1038/nature13527. http://dx.doi.org/10.1038/ nature13527. Published Version doi:10.1038/nature13527 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:16121077 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA HHS Public Access Author manuscript Author Manuscript Author ManuscriptNature. Author ManuscriptAuthor manuscript; Author Manuscript available in PMC 2015 May 07. Published in final edited form as: Nature. 2014 August 7; 512(7512): 49–53. doi:10.1038/nature13527. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide Eric S. Fischer1,2, Kerstin Böhm1,2, John R. Lydeard3, Haidi Yang5, Michael B. Stadler1,2,6, Simone Cavadini1,2, Jane Nagel5, Fabrizio Serluca5, Vincent Acker4, Gondichatnahalli M. Lingaraju1,2, Ritesh B. Tichkule5, Michael Schebesta5, William C. Forrester5, Markus Schirle5, Ulrich Hassiepen4, Johannes Ottl4, Marc Hild5, Rohan E. J. Beckwith5, J. Wade Harper3, Jeremy L. Jenkins5, and Nicolas H. Thomä1,2,* 1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland 2University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland 3Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston MA 02115, USA 4Novartis Pharma AG, Institutes for Biomedical Research, Novartis Campus, CH-4056 Basel, Switzerland 5Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge MA 02139, USA 6Swiss Institute of Bioinformatics, Maulbeerstrasse 66, CH-4058 Basel, Switzerland Abstract In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. *Correspondence and requests for materials should be addressed to [email protected]. Supplementary information is linked to the online version of the paper at www.nature.com/nature. Author Contributions E.S.F., N.H.T., J.L.J and W.C.F initiated the project. E.S.F. and K.B. conducted the protein purification and crystallisation. S.C. pre-screened protein complexes by EM. E.S.F collected data, processed and refined x-ray data. E.S.F. and N.H.T. analysed the structures. E.S.F. performed in vitro experiments and, with the help of U.H., developed and performed TR-FRET and FP assays. E.S.F. performed protein array experiments, M.B.S. and E.S.F. analysed the data. E.S.F., K.B., J.R.L., H.Y., M.H., J.W.H. and N.H.T. conceived and performed the cell biological characterisation. R.B.T. and R.E.J.B. conceived and conducted chemical syntheses. J.N. and M.S. performed proteomics. V.A. and J.O. did the DSF experiments. F.S. and M.S. did the zebrafish experiments. E.S.F. and N.H.T. wrote the manuscript. All authors assisted in editing the manuscript. Author Information The following structural coordinates have been reported and were submitted to the Protein Data Bank under accession numbers: hsDDB1-ggCRBN-thalidomide (pdb:4CI1), hsDDB1-ggCRBN-lenalidomide (pdb:4CI2), hsDDB1-ggCRBN- pomalidomide (pdb:4CI3). Human protein microarray data sets generated for this study are available from GEO (http:// www.ncbi.nlm.nih.gov/geo) under accession GSE57554. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Fischer et al. Page 2 This dual activity implies that small molecules can principally modulate a ligase to up- or down- Author Manuscript Author Manuscript Author Manuscript Author Manuscript regulate the ubiquitination of proteins. Thalidomide (α-(N-phthalimido)glutarimide) was introduced to the market in 1954 by Chemie Grünenthal. Initially promoted as a sedative with anti-emetic properties1,2, it became popular in the treatment of ‘morning sickness’3. In 1961, thalidomide taken in the first trimester of pregnancy was implicated in frequent limb deformities in infants4,5. Between 8,000 and 12,000 affected children were born before the drug was banned. Interest in thalidomide revived in 1965 when it was shown to have immunomodulatory and anti- inflammatory properties in erythema nodosum leprosum (ENL), an inflammatory complication of leprosy6. In 1994, thalidomide was found to inhibit fibroblast growth factor (bFGF)-induced formation of new blood vessels7. These findings prompted clinical trials exploring thalidomide use in anti-angiogenic cancer therapy. The efficacy of thalidomide and its derivatives lenalidomide and pomalidomide (collectively known as IMiDs) has since been demonstrated in several blood cancers8 : newly diagnosed multiple myeloma (thalidomide)9, refractory multiple myeloma (lenalidomide/pomalidomide) and 5q-deletion- associated myelodysplastic syndrome (lenalidomide). The target of thalidomide, cereblon (CRBN), is a ubiquitously expressed protein that is part of the cullin-4 E3 ubiquitin ligase complex, CUL4-RBX1-DDB1 (CRL4)10. Mutations in CRBN are associated with autosomal recessive non-syndromic mental retardation (MR)11. In myeloma cells, the anti-proliferative activities of IMiDs are linked to CRBN expression12,13, making IMiDs the first clinically approved E3 ubiquitin ligase inhibitors with specificity for the CRL4CRBN ligase12. The IMiD anti-proliferative and immunomodulatory effects have recently been linked to drug-induced ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors by CRL4CRBN14 –16. Accordingly, loss of CRBN is a common determinant of drug resistance in myeloma cells12. How IMiD binding affects the CRL4CRBN ligase at the molecular level remains unclear. We set out to examine the role of CRBN within the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3-ligase complex, characterising the effect of IMiD binding on ligase activity. Structure of DDB1-CRBN bound to IMiDs We crystallized a chimeric complex of human DDB1 (DDB1) and chicken CRBN (ggCRBN) bound to thalidomide (refined to 3.0 Å), lenalidomide (3.0 Å) and pomalidomide (3.5 Å) (Fig. 1a, b and Extended Data Table 1). The high level of sequence conservation between human and chicken CRBN (Extended Data Fig. 1a, b) allows structural insights to be inferred directly from chicken to human CRBN. All subsequent biochemical and cell- biological experiments were performed with human full-length proteins. ggCRBN consists of three sub-domains (Extended Data Fig. 2a–f): a seven-stranded β-sheet located in the N- terminal domain (NTD, residues 1–185) (Extended Data Fig. 2a), a 7-α-helical bundle domain (HBD, residues 186–317) involved in DDB1 binding (Fig. 1c), and a C-terminal domain composed of 8 β-sheets (CTD, residues 318–445) (Fig. 1b). DDB1 comprises three seven-bladed WD40 β-propellers arranged in a triangular fashion (BPA, BPB and BPC)17 with ggCRBN attaching to a cavity between the BPA and BPC propellers (Fig. 1c). The molecular basis of the HBD-mediated attachment of ggCRBN to DDB1 defines a novel Nature. Author manuscript; available in PMC 2015 May 07. Fischer et al. Page 3 class of DDB1 binders and differs in detail from previous DDB1 attachment modules17 –20 Author Manuscript Author Manuscript Author Manuscript Author Manuscript (Extended Data Fig. 2e, f). The ggCRBN N-terminal region (residues 46–317) including the NTD and HBD resembles the N-terminal domain of bacterial Lon proteases (PDB: 3LJC - RMSD of 2.7 Å over 178 residues aligned) (Extended Data Fig. 2b). The CTD harbours the thalidomide-binding pocket and contains a conserved Zn2+-binding site situated approximately 18 Å from the compound (Fig. 1a, b). The Zn2+ ion is coordinated through conserved cysteine residues 325, 328, 393 and 396. The ggCRBN-CTD shares structural similarity with the pseudouridine synthase and archaeosine transglycosylase (PUA) fold family21 involved in the binding of diverse sets of ligands (Extended Data Fig. 2c, d). IMiD binding to CRBN Thalidomide, lenalidomide and pomalidomide (Fig. 2a–c and Extended Data Fig. 3a–i) bind a pocket on the ggCRBN-CTD (Fig. 1b) situated in a surface groove that is highly conserved across CRBN orthologues (Extended Data Fig. 1b). The three ligands superimpose with very little deviation in the α-(isoindolinone-2-yl) glutarimide moiety, which contributes the majority of interactions between the receptor and the compounds and represents the main pharmacophore22. The glutarimide group is held in a buried cavity between ggCRBN sheets β10 and β13(Fig. 2d). Glutarimide carbonyls (C2, C6) and the intervening amide (N1) are in hydrogen-bonding distance to ggCRBN residues His380 and Trp382, respectively (Fig. 2c, d). A delocalised lone pair connects the glutarimide nitrogen with the two glutarimide carbonyls (C2-N1-C6) and is coplanar with Trp382. The opposing aliphatic face of the glutarimide ring (C3, C4 and C5) is in tight Van-der-Waals contact with a hydrophobic pocket lined by Trp382, Trp388, Trp402 and Phe404.